Recombinant DNA technology has advanced to the point where the cloning of select DNA sequences has become possible. According to well known procedures various cohesive DNA fragments can be ligated in vitro to produce powerful expression vectors capable of transforming host organisms to express polypeptide sequences foreign to the host cell. See, Maniatis, T. et al., Molecular Cloning, Cold Spring Harbor Laboratory (1982); Cohen et al., U.S. Pat. No. 4,237,224. A generalized discussion of the subject matter appears in S. Cohen, Scientific American, 233, 24 (1975). These and other publications alluded to herein are incorporated by reference.
Generally, expression vectors contain DNA encoding a desired polypeptide, an origin of replication, one or more phenotypic selection characteristics and an expression promoter. The expression vector is introduced into its host cells by a process called "transformation" and large quantities of the expression vector are obtained by growing the transformed cells. Where DNA encoding for the desired polypeptide is inserted within reading relationship to the promoter, the mechanisms for the biosyntheses of protein within the host cell transcribe and translate the DNA into polypeptide in a two-step process referred to as "expression."
In the first step, the DNA is enzymatically transcribed in the form of messenger RNA (mRNA) by RNA polymerase. The transcription of DNA is initiated in a region known as the promoter. The promoter contains the RNA polymerase recognition site where the promoter is recognized and bound by RNA polymerase. The promoter sequence itself generally consists of a sequence rich in adeninethymidine base pairs (i.e., TATA) and is not transcribed. Also located 5' to the AT rich sequence is a CAAT sequence which may also be involved in transcription initiation. For a review, see Kessel, M. and Khoury, G., Gene Amplification and Analysis, Vol. 3, Papas et al., ed. New York, Elsevier/North Holland, Inc. 1983, pp. 234-266.
Soon after binding to RNA polymerase recognition site, RNA polymerase begins to diffuse down the DNA sequence. Eventually the RNA polymerase will encounter the transcription start site at which point the RNA polymerase begins to transcribe mRNA from the DNA sequence coding for the polypeptide. The DNA sequence encoding the polypeptide is referred to as the "structural gene". In the second step, the mRNA is translated into a polypeptide having the amino acid sequence for which the structural gene codes.
Expression of certain polypeptides is regulated at the point of transcription by certain "regulatory sequences." In procaryotic systems, expression of certain sets of genes has been shown to be regulated by operator, promoter and attenuator sequences. These sequences are located at the 5' end of the structural genes. For example, the lactose operon of E. coli contains an operator. An operator is a DNA sequence which is recognized by repressor proteins. The repressor binds to the operator and thereby sterically prevents RNA polymerase from binding to the adjacent promoter or diffusing down the DNA sequence. The repressor therefore has the ability to shut off expression of the particular gene. A substance called the "inducer" deactivates the repressor, freeing the operator and permitting RNA polymerase to bind to the promoter.
The tryptophan (trp) operon of E. coli is another example of an inducible system. The trp operon is repressed by tryptophan, which binds to the specific repressor and thereby enables it to interact with the operator. The effect is to switch off the transcription of the genes that code for the biosynthesis of tryptophan. Chang et al., Nature, 275, 615 (1978); Itakura, et al., Science, 198 (1977); Goeddel, et al., Nucleic Acids Res., 8, 4057 (1979). Several operons for the biosynthesis of amino acids, including the trp operon, are also controlled by an attenuator site. Transcription terminates at this site if the amino acid end product is abundant. Jacob and Monod, J. Mol. Biol., 3, 318-356 (1961); Miller and Reznikoff, The Operon, Cold Spring Harbor Laboratory (1978); Chang et al., Nature, 275, 615 (1978).
Inducible systems are also found in eukaryotics. One example is the yeast repressible acid phosphatase (A. Pase) gene, pH05. Schurr and Yagil, J. Gen. Microbiol., 65, 291-303 (1971). Transcription of the pH05 gene is tightly repressed when inorganic phosphate is present in the growth medium but is induced to high levels when inorganic phosphate is depleted. Bostian et al., Proc. Natl. Acad. Sci. U.S.A., 77. 4504-4508 (1980).
The regulatory/promoter sequences of these procaryotic operon systems as well as the yeast systems have been introduced into expression vectors to direct the synthesis of polypeptides under induced and non-induced conditions in E. Coli host cells or yeast saccharomvces cerevisiae systems. See, e.g., Kramer, R. A. et al., Proc. Natl. Acad. Sci. U.S.A., 81, 367-370 (1984). These regulatory/promoter sequences, although useful in procaryotes and lower eucaryotes such as the yeast, are not always applicable for higher eukaryotic host systems, such as mammalian tissue culture cells.
In recent years, the availability of cloned genes and methods by which they are transferred into eukaryotic cells, has made it possible to identify similar regulatory sequences in animal cells. For purposes of the present invention, the term "regulatory sequences" refers to the regulatory sequences which are required for transcription regulation of a gene system and may or may not include a promoter sequence.
The experimental approach towards the investigation of such regulatory sequences falls into two categories. In both cases, hybrid genes consisting of a putative control region from the inducible gene and the structural gene sequences coding for drug resistance or a enzymatic activity are constructed. In one approach, these hybrid genes are transfected into tissue culture cells and single-cell clones which have integrated these hybrid genes into their chromosomes are grown up under selective conditions. To demonstrate the inducibility of these integrated genes, expression in the presence and absence of the inducible agent is evaluated by RNA analysis or by direct measurement of the protein product of the inducible gene. In the second approach, the hybrid gene is simply introduced into the eukaryotic cell of interest. Shortly after transfection (generally several hours to a few days) an extract is made of the cultures, which have been maintained in the presence or absence of inducer. The extract is then analyzed for the gene products (RNA or protein).
These methods have been used successfully to locate specific eukaryotic regulatory sequences. Examples are mouse and human metallothionein genes which are inducible by heavy metals [Mayo et al., Cell, 29, 99-108 (1982), Karin et al., Cell, 36, 371-379 (1984)]and the Drosophila heat shock regulatory sequence which is inducible by heat [Pelham, Cell, 30, 517-528 (1982)]. Other regulatory sequences are isolated from viral sources such as the SV40 early promoter/enhancer and that of the mouse mammary tumor viruses. Lee F., et al., Nature, 294, 228-232 (1981).
Inducible regulatory sequences are useful in the area of recombinant DNA technology. These regulatory sequences can be inserted into recombinant DNA expression vectors for the controlled expression of a desired gene. Inducible regulatory sequences are particularly useful when the desired polypeptide is toxic to the host cell. When the polypeptide is toxic to the host cell the cell will be unable to replicate in any significant amount. An inducible regulatory sequence can be used to shut off expression of the toxic polypeptide until the host cells are replicated. Once a desired amount of host cells are obtained the regulatory sequence can be activated by varying culture conditions, thus enabling large quantities of the peptide to be produced.
The regulatory sequence of the present invention is another example of a mammalian regulatory sequence. It has the unique ability to enhance transcription under conditions of glucose starvation or calcium shock in a variety of eukaryotic host systems. The regulatory sequence can also enhance transcription by high temperature when introduced into the temperature-sensitive Chinese hamster cell line K12 or other systems having a compatible mutation.